Abstract The enzyme poly(ADP-ribose)glycohydrolase (PARG) is critical for the response of cells to DNA damage, and genetic and biochemical evidence indicates that PARG inhibition is a tantalizing strategy for the treatment of cancer and possibly other disease. Unfortunately, no potent, selective, and cell permeable inhibitors of PARG exist, thus there are no suitable small molecule probes to study the cellular function of this enzyme and to examine its therapeutic potential. The lack of suitable PARG inhibitors is directly attributable to the lack of an assay that can be used to monitor PARG enzymatic activity in a high-throughput fashion; the standard PARG enzymatic assay uses radiolabeled poly(ADP-ribose) and thin-layer chromatography. We have recently developed a novel PARG substrate in which a chromophore is liberated by PARG-mediated cleavage. Using this substrate, we have now conducted pilot screens in 384-well plates using liquid handling robots and full-length human PARG, and these experiments show this assay to be robust and excellent for the identification of PARG inhibitors. We now propose (in Specific Aim 1) to use this novel assay to screen ~200,000 compounds at the University of Illinois High- Throughput Screening Facility (HTSF). As described in Specific Aim 2, we have developed a critical path for follow-up experiments based on a tiered series of increasingly stringent secondary assays, both in vitro and cell culture analyses. Our goal by the end of the two-year funding period is to have identified a potent, selective, and cell permeable PARG inhibitor that can be widely used by the community for the interrogation of PARG activity in cells and for probing the potential of PARG inhibition for the treatment of various disease.